Studies on DNA replication of plasmid ColEl and its relatives have been continued. Transcripts by RNA polymerase (RNA II) that start the 555 nucleotides upstream of the replication origin of plasmid ColEl extend beyond the origin. The hybridized transcript is cleaved by ribonuclease H at the origin and used as the primer for DNA synthesis by DNA polymerase I. The primer formation is dependent on the secondary structure of RNA II. A single base change far from the replication origin can affect a specific stage of primer formation. Thus each single base-change affects initiation of synthesis of RNA II, hybrid formation between RNA II and the template DNA, the cleavage sites of RNA II by RNase H, the stability of association of RNA II or the use of cleaved RNA II as a primer by DNA polymerase I. Supressor mutations that restore the activity of RNA II lost by a mutation have been found. Based on the results of analysis of mutant RNA IIs by partial digestion with an RNase we could deduce the structure of functional RNA II. Primer formation is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to RNA II at the complementary region. This binding results in inhibition of formation of the secondary structure necessary for primer formation. Based on the results of kinetic analysis of binding of RNA I to RNA II, we proposed a stepwise model of binding of RNA I to RNA II. Primer formation is also regulated by a protein specified from a region downstream of the replication origin. This 63-amino acid protein stimulates binding of RNA I to RNA II and thus enhances the inhibitory action of RNA I. Inhibition of primer formation by RNA I in the presence or absence of the protein determines the copy number of a plasmid in a cell and the incompatibility between related plasmids.